Intraepithelial lymphocytes (IELs) from the small intestine constitute a heterogeneous population of T cells that form the first line of adaptive immune defense at the body's mucosa, the main entrance for pathogens. Although IELs constitute one of the largest T cell compartments of the body our understanding of their development and function is particularly limited. In recent years, our lab has made some of the most advanced and significant insights into the development of CD8?? TCR?? IELs, showing that they are generated in the thymus and that their precursor thymocytes undergo a self-agonist dependent selection also called agonist selection. These observations were made using commonly available TCR/cognate antigen dual transgenic mouse systems. Unfortunately, all of these models used TCRs derived from conventionally selected CD4 or CD8?? T cells that recognize known antigens. It is likely that the TCR repertoire of conventional T cells, which are mostly devoid of autoreactive specificities, differs from the oligoclonal and possibly self-reactiv repertoire of CD8?? TCR?? IELs. Therefore we believe that these transgenic models are not appropriate for further analysis and characterization of agonist selection and development of CD8?? TCR?? IELs. Here we propose an original approach aiming to elucidate the MHC restriction and providing new insights into differentiation pathway of CD8?? TCR?? IELs. First, we will generated a TCR repertoire sequence database from CD8?? TCR?? IELs using massively parallel sequencing. IEL will be isolated from multiple non-pooled mice expressing a fixed TCR? (TCR V?? Tg model); thus each individual TCR? chain sequences obtained by High Throughput sequencing will represent a complete clone when paired with the unique TCR V?5 chain. TCR clones that are highly represented within the repertoire will then be cloned and expressed retrovirally in different MHC-deficient recipients in order to assess and identify the restricting MHC molecules (Aim 1). This first approach will allow us rapidly and efficiently to screen a number of potential TCR clones that will subsequently be used to address the differentiation pathway of CD8?? TCR?? IELs. We will do this using a retroviral system that drives the temporally-regulated expression of the TCR? chain. With this approach, TCR? chains will start to be expressed specifically in CD4- expressing double positive thymocytes using a Cre-regulated retroviral vector. Analysis of the chimera should allow us to give a detail description of the differentiation pathway of CD8?? TCR?? IELs in vivo (Aim 2). Both aims are complementary and will lead to a) unravel the MHC restriction of CD8?? TCR?? IELs and b) provide with new insight regarding into the thymic development of agonistically selected CD8?? TCR?? IELs. Completion of this study will provide us with a tremendous amount of new and important information regarding the biology of CD8?? TCR?? IELs. A better understanding of the intestinal immune system holds promise of the development of novel therapeutic modalities for a number of intestinal inflammatory disorders. PUBLIC HEALTH RELEVANCE: Given the profound role the intestinal immune system plays in generation of tolerance and protective immunity, it is surprising how little the potential of this system has been utilized in clinical practice. Unconventional intraepithelial lymphocytes constitute a substantial population of T cells in the intestine, yet their role remains elusive. Hee we set out to explain how they differentiate and how they recognize foreign molecules, information that is critical in understanding how the intestinal immune lymphocytes are capable of eliciting an immune response against pathogens.